This invention relates to a DNA hybridization incubation chamber for use in performing DNA hybridization assays.
High density arrays are new tools used by drug researchers and geneticists which provide information on the expression of genes from particular cells. A high density array typically comprises between 5,000 and 50,000 probes in the form of DNA strands, each of known and different sequence, arranged in a determined pattern on a substrate. The substrate may be any size but typically takes the form of a 1xc3x973 inch glass microscope slide.
The arrays are used to determine whether target sequences interact or hybridize with any of the probes on the array. After exposing the array to target sequences under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has hybridized with the probe at that location. DNA arrays can be used to study which genes are xe2x80x9cturned onxe2x80x9d or up regulated and which genes are xe2x80x9cturned offxe2x80x9d or down regulated. So for example, a researcher can compare a normal colon cell with a malignant colon cell and thereby determine which genes are being expressed or not expressed only in the aberrant cell. The regulation of these genes serves as key targets for drug therapy.
Hybridization is a hydrogen bonding interaction between two nucleic acid strands that obey the Watson-Crick complementary rules. All other base pairs are mismatches that destabilize hybrids. Since a single mismatch decreases the melting temperature of a hybrid by up to 10 degrees C., conditions can be found at which only perfect hybrids can survive. Hybridization comprises contacting the strands, one of which is immobilized on the substrate and the other which usually bears a radioactive, chemoluminescent or fluorescent label, and then separating the resulting hybrids from the unreacted labeled strands by washing the support. Hybrids are recognized by detecting the label bound to the surface of the support.
In performing the hybridization, depending on reagent (buffer) compositions employed, and the similarity of the probe and target molecules, the temperature employed may vary from about ambient temperature to about 70xc2x0 C. As described, temperature is used as a process variable in altering the hybridization stringency. Typically, nucleic acid and protein hybridizations are carried out in a closed container in a constant temperature environment for extended periods of time, e.g., 10-18 hours.
Since the hybridization assays require tight temperature control and a controlled environment, researchers use an enclosed system, often referred to as a hybridization chamber, in order to perform hybridization assay. The standard hybridization chamber consists of a plastic (typically polypropylene) two-piece construction. A base portion and a top portion join together to define an internal sealed chamber. The chamber is environmentally sealed by a rubber o-ring gasket assembly which both prevents ambient moisture or air from entering the chamber, as well as the escape of any liquid or vapor from the sample itself out of the chamber. The unit is completely sealed by the use of an o ring and external clamps.
The substrate, which contains the tethered array of probe nucleotides, is placed in the chamber. A small amount or minimal amount buffer solution containing the target probes is deposited on the array and is spread and covered with a cover-slip. The chamber is closed and sealed with the clamp mechanism, and the entire chamber is introduced into a temperature controlled environment in the form of a water bath, conventional oven, or hybridization incubator for example.
It has been discovered that incubating samples in the standard hybridization chamber at elevated temperature causes the sample at the edges of the cover-slip to evaporate into the cavity of the chamber. This evaporation causes the sample to dry out around the edges of the cover-slip. In turn, it has been found that hybridization either does not occur in these dried out areas, or is severely compromised.
By providing a liquid filled reservoir within the sealed environment of the hybridization chamber, the present invention solves the problem of excessive drying of the sample. The liquid in the reservoir evaporates into the environment of the sealed chamber thereby saturating the air and thus preventing the drying phenomenon around the edges of the cover-slip.
The present invention provides a hybridization chamber that contains a built-in mechanism for saturating the air within the chamber when sealed thereby preventing drying of the liquid sample. The hybridization chamber is defined by matching top and bottom clam-shell like halves that, when brought together, are sealed by an o-ring and clamping device. The chamber is equipped with a liquid reservoir, the liquid from which will serve to saturate the volume of air sealed within the hybridization chamber. A saturated atmosphere within the chamber prevents evaporation of the sample.